Evaluating proteins

ABSTRACT

The disclosure features methods that include: providing a substrate that includes (i) a nucleic acid (e.g., DNA or RNA) encoding a hybrid amino acid sequence including a test amino acid sequence and an affinity tag, and (ii) a binding agent that recognizes the affinity tag; contacting the substrate with a translation effector to thereby translate the hybrid amino acid sequence; maintaining the substrate under conditions permissive for the hybrid amino acid sequence to bind the binding agent; and removing the nucleic acid from the substrate. In one embodiment, the substrate includes a plurality of positionally-distinguishable addresses, for example, each includes a different nucleic acid. The addresses can be located a regularly or irregularly spaced locations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/770,111, filed on Jun. 28, 2007, which claims the benefit of thefiling date of U.S. Provisional Application Nos. 60/806,245, 60/806,252,and 60/806,253, which were filed on Jun. 29, 2006. The contents of U.S.application Ser. No. 11/770,111 and U.S. Provisional Application Nos.60/806,245, 60/806,252, and 60/806,253 are incorporated by reference aspart of this application.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has certain rights in this invention pursuant toGrant No. CA099191/CA117374 awarded by National Cancer Institute andContract No. HHSN266200400053 awarded by National Institute of Allergyand Infectious Diseases.

BACKGROUND

Protein array technology has facilitated the high-throughput analysis ofproteins, including the large-scale characterization of numerous bindinginteractions and enzymatic activities. Protein arrays can be made by avariety of methods including depositing proteins onto an array surfaceand translating proteins on the array.

SUMMARY

In one aspect, this disclosure features a method that includes:providing a substrate that includes (i) a nucleic acid (e.g., DNA orRNA) encoding a hybrid amino acid sequence including a test amino acidsequence and an affinity tag, and (ii) a binding agent that recognizesthe affinity tag; contacting the substrate with a translation effectorto thereby translate the hybrid amino acid sequence; maintaining thesubstrate under conditions permissive for the hybrid amino acid sequenceto bind the binding agent; and removing the nucleic acid from thesubstrate. In one embodiment, the substrate includes a plurality ofpositionally-distinguishable addresses, for example, each include adifferent nucleic acid. The addresses can be located a regularly orirregularly spaced locations.

In one embodiment, removing the nucleic acid includes digesting thenucleic acid with an enzyme, e.g., an RNase or DNase.

The nucleic acid can be attached to the substrate, e.g., non-covalentlyattached by a reagent whose affinity for nucleic acid varies with bufferconditions. For example, the reagent binds nucleic acid at a pH of lessthan 7.5, but releases nucleic acid at a pH greater than 8. An exemplaryreagent is mono-amino-N-aminoethyl (MANAE).

In one aspect, the disclosure features a substrate that includes asurface that includes a surface modification with a group thatselectively binds to nucleic acid, and coding nucleic acids distributedat various locations on the surface. For example, the substrate issubstantially planar. In one embodiment, the surface further includes abinding agent that recognizes a tag and the coding nucleic acids encodepolypeptides that include the tag. In one embodiment, the surfacemodification is mono-amino-N-aminoethyl (MANAE). The substrate canfurther include a non-protein agent that binds to the Fc region of anantibody (e.g. a boronate group, or a metal chelating group)

In another aspect, the disclosure features method that includes:providing a substrate that includes a reactive surface having ahomo-functional group capable of reacting with proteins (e.g., a primaryamino group, an aldehyde group, an epoxy group, or a carboxyl group);and disposing, at a plurality of positionally distinguishable locationson the reactive surface, (i) a nucleic acid encoding a hybrid amino acidsequence including a test amino acid sequence and an affinity tag, and(ii) a binding agent that recognizes the affinity tag. The binding agentreacts with the reactive surface and becomes covalently attached to thesurface. The nucleic acid and the binding agent can be disposed on thesubstrate separately or together. The method can further includeinactivating the reactive surface and contacting the substrate with atranslation effector to thereby translate the hybrid amino acid sequenceat each of the locations; and maintaining the substrate under conditionspermissive for the hybrid amino acid sequence to bind the binding agent.

In another aspect, the disclosure features a substrate having a surfacemodified with a non-protein agent that binds to the Fc region of anantibody (e.g. a boronate group, a metal chelating group, or animmobilized iminodiacetic acid (IDA) chelated to a metal, e.g.,IDA-copper), wherein the substrate includes a plurality of differentcoding nucleic acids disposed at different locations on the surface. Forexample the substrate is an array. The substrate can include one or moreproteins that include an Fc region bound to the non-protein agent and/orone or more antibodies that include an Fc region bound to thenon-protein agent.

In still another aspect, the disclosure features a substrate including asurface modified with heterofunctional groups, including at least onegroup that physically absorbs proteins and at least one group that cancovalently attach proteins, wherein the substrate includes a pluralityof different coding nucleic acids disposed at different locations on thesurface. For example, at least one of the groups can promote thephysical adsorption of proteins by ionic exchange, by phenyl-boronicadsorption, or by IMAC adsorption. In some embodiments, at least one ofthe groups is an epoxy group. In some embodiments, the surface includesepoxy and amino groups; amino and phenyl-boronic groups; or amino andIMAC groups, or combinations thereof.

In yet another aspect, the disclosure features a substrate that includesa surface modified with a non-protein agent that binds to the Fc regionof an antibody, and a reagent whose affinity for nucleic acid varieswith buffer conditions, wherein the substrate comprises a plurality ofdifferent coding nucleic acids disposed at different locations on thesurface.

In another aspect, the disclosure features a method that includes: (a)providing the substrate having a surface modified with a non-proteinagent that binds to the Fc region of an antibody (e.g. a boronate group,a metal chelating group, or an immobilized iminodiacetic acid (IDA)chelated to a metal, e.g., IDA-copper), wherein the substrate includes aplurality of different coding nucleic acids disposed at differentlocations on the surface; and (b) translating the nucleic acids of thesubstrate by contacting the substrate with a transcription effector.

In another aspect, this disclosure features a substrate that includes:(a) a metallic surface (e.g., a gold surface); and (b) an array ofprotein aggregates disposed on the surface. In some embodiments, thesurface is substantially planar. In other embodiments, the surface isnon-planar. For example, the aggregates are formed by crosslinkingsoluble proteins, e.g., prior to disposing the proteins on the surface,during or after disposing the proteins on the surface.

This disclosure also features a method of providing a protein array. Themethod includes: providing a plurality of protein samples; formingprotein aggregates in each of the protein samples; and disposing theprotein aggregates on a metallic surface (e.g., a gold surface), e.g.,at positionally distinguishable locations on the metallic surface. Insome embodiments, the samples include soluble proteins. In someembodiments, the protein aggregates are formed by addition of acrosslinker, e.g., a homo-bifunctional crosslinker or ahetero-bifunctional crosslinker. In some embodiments, the metallicsurface is substantially planar.

In another aspect, the disclosure features a method that includesproviding a substrate that includes (i) a nucleic acid encoding a hybridamino acid sequence including a test amino acid sequence and an affinitytag wherein the amino acid sequence includes a transmembrane domain, and(ii) a binding agent that recognizes the affinity tag; contacting thesubstrate with a translation effector to thereby translate the hybridamino acid sequence; and maintaining the substrate under conditionspermissive for the hybrid amino acid sequence to bind the binding agent,thereby attaching a protein that has a transmembrane domain to thesubstrate. The amino acid sequence can include at least two, three, orfour transmembrane domains, e.g., seven transmembrane domains.

For example, at least some of the amino acid sequence include amammalian receptor polypeptide or a fragment thereof. For example, atleast some of the amino acid sequences include cytokine or growth factorreceptors or fragments thereof.

The disclosure also features a substrate that includes a plurality ofpositionally distinguishable locations, wherein different proteins areattached to the substrate at the different locations, at least some ofthe proteins including a transmembrane domain. For example, at leastsome of the proteins are mammalian receptor polypeptides or includefragments thereof. At least some of the proteins can include at leasttwo transmembrane domains, three, or four transmembrane domains, e.g.,seven transmembrane domains.

In some embodiments, at least some of the proteins are translated on thesubstrate. In some embodiments, the substrate has at least 10, 50, 100,or 500 different proteins that include a transmembrane domain attachedto it and each of these proteins is at a different positionallydistinguishable location.

In another aspect, the disclosure features a method for evaluating aplurality of proteins. The method includes: providing a substrate havinga plurality of different proteins attached at different positionallydistinguishable locations contacting each of the locations with a cell,to provide a substrate having cells contacted thereto; and evaluatingeach location. For example, the cells are mammalian cells, e.g., immunecells (e.g., T cells or B cells), fibroblasts, stem cells, or neuronalcells.

In some embodiments, each location includes a reagent for evaluating acell; and the method includes detecting the reagent at each of thelocations to evaluate the effect of the respective protein at eachlocation on the cell at that location, thereby evaluating a plurality ofproteins. For example, the reagent is an antibody or other specificbinding protein that can bind to a secreted agent, e.g., a secretedprotein such as a cytokine or growth factor, e.g., interferon gamma.

In some embodiments, the cells include a image-detectable reagent, e.g.,a fluorescent protein or an environment-sensitive dye, e.g., a calciumsensitive dye.

At least some of the proteins can include a transmembrane domain.

In another aspect, the disclosure features a method for evaluating aplurality of proteins. The method includes: providing a substrate havinga plurality of different proteins attached at different positionallydistinguishable locations; contacting each of the locations with a cell,to provide a substrate having cells contacted thereto; and evaluatingthe cell at each of the locations, thereby evaluating a plurality ofproteins.

In some embodiments the cells include a image-detectable reagent, e.g.,a fluorescent protein, and a calcium-sensitive dye.

In some embodiments, at least some of the proteins include atransmembrane domain.

In another aspect, the disclosure features a substrate including aplurality of positionally distinguishable locations, wherein differentproteins are located on the substrate at the different locations, eachlocation includes a cell that can interact with the protein at thatparticular location, and each location includes a reagent evaluating theeffect of the respective protein at each location on the cell at thatlocation.

In some embodiments the cells are immune cells, e.g., T cells or Bcells. In some embodiments the reagent is an antibody that can bind to asubstance, e.g., interferon gamma, secreted by the cells.

In another aspect, this disclosure features a substrate that includes:(a) a surface that is modified with a poly-blocker with multiplefunctional groups; and (b) an array of macromolecules (e.g., nucleicacids or proteins) disposed on the surface. For example, thepoly-blocker includes a polyanion, e.g., a sulfate-dextran, acarboxymethyl-dextran, an aspartic-dextran, a glutamic dextran or acombination thereof. Combinations of poly-anions can also be used, e.g.,a combination of large and small molecular weight polyanions. Forexample, the poly-anion can be soluble at mild conditions. It is alsopossible to use linkers with different sources of negative charges,e.g., carboxyls or phosphates. In some embodiments, linkers with otherfunctional groups such as aldehydes or epoxys can be used for blocking.In some embodiments, the poly-blocker is covalently bound to thesurface; in others it is non-covalently bound to the surface. Thepoly-blocker can range in molecular weight. For example, the averagemolecular weight of the poly-blocker can be, e.g., between 1-10 kDa, orhigher. Higher molecular weight polyblockers, e.g., polyanions, can beused with lower density of the amino groups.

The surface can be positively-charged and the poly-blocker isnegatively-charged. For example, the surface is poly-lysine orpoly-arginine.

The surface can be negatively-charged and the poly-blocker ispositively-charged.

In some embodiments, the surface includes aldehyde groups and thepoly-blocker includes amino groups, e.g., primary aminos that react withthe aldehyde groups.

In some embodiments, the surface includes activated ester groups and thepoly-blocker includes amino groups that react with the ester groups.

In some embodiments, the surface includes an amino moiety (e.g., primaryor secondary amino group) and the poly-blocker is a sulfate-dextran, acarboxymethyl-dextran, an aspartic-dextran, a glutamic-dextran or acombination thereof.

In some embodiments, the substrate can be glass, e.g., a glass slide.The substrate can be planar or non-planar. The substrate an includeinvaginations or pores.

In some embodiments, the surface can include poly-lysine orpoly-arginine. The surface can include aldehyde groups and thepoly-blocker can include amine groups, e.g., primary amine groups, thatcan react with the aldehyde groups. The surface can include activatedester groups and the poly-blocker can include amine groups that canreact with the ester groups.

The substrate can include other features described herein.

In another aspect, the disclosure features a method that includes:

(a) providing a substrate including a surface having a plurality ofaddresses;(b) disposing a plurality of nucleic acids that each include a codingregion at addresses on the surface; and (c) adhering a poly-blocker withmultiple functional groups to the surface. Step (c) can precede step (b)or follow step (c). For example, a different nucleic acid is disposed atthe different addresses. Some addresses can be duplicates of otheraddresses.

In some implementations each nucleic acid of the plurality includes ananchoring agent that anchors the nucleic acid to the surface.

The coding region of each nucleic acid of the plurality encodes apolypeptide that includes a first amino acid sequence and an affinitytag. Each address can include a binding agent that recognizes theaffinity tag.

The method can further include translating each nucleic acid of theplurality with a transcription and a translation effector. In manyembodiments, step (c) is completed before the translating.

The method can include other features described herein.

In another aspect, the disclosure features a method that includes:

(a) providing a substrate including a surface having a plurality ofaddresses;(b) disposing a plurality of proteins at addresses on the surface; and(c) adhering a poly-blocker with multiple functional groups to thesurface. Step (c) can precede step (b) or follow step (c). Thepoly-blocker can regulate stringency of interactions between surfaceproteins and interacting agents. The method can include other featuresdescribed herein.

The term “protein” refers to peptides, polypeptides, and assemblies ofmore than one polypeptide chains (e.g., multi-subunit proteins).

Protein arrays have a variety of applications. They can be used toprovide a diagnosis for a subject. For example, a sample from thesubject can be contacted to the array to evaluate a property of thesample. (See, e.g., US 2005/0048580). Protein arrays can be used toevaluate one or more test compounds, e.g., to identify drug candidates.(See, e.g., US 2005/0048580). Arrays can be used to examine targetprotein interactions with other molecules, such as drugs, antibodies,nucleic acids, lipids, or other proteins. In addition, the array can beinterrogated to find substrates and cofactors for enzymes.

The methods described herein can be adapted to a variety of formats. Forexample, they can used to provide an arrayed collection of ligands,e.g., specific antibodies that can measure the presence and abundance ofspecific proteins (or other molecules). They can be used to provide anarrayed collection of any protein of interest, or sets of proteins, forexample, to study protein function (e.g., an activity such as binding orcatalytic activity), drug interactions, and protein-proteininteractions. The arrayed proteins can be artificial variants of aparticular protein (e.g., a given natural protein). The artificialvariants can be produced by targeted mutagenesis or random mutagenesis.The arrayed proteins can be related by class or function, e.g., cellsurface receptors, proteases, adhesion proteins, and so forth.

The methods and surfaces described herein can also be used in non-arrayapplications, e.g., to modify other surfaces, e.g., beads, membranes,apparati and other device parts.

The methods and surfaces described herein include, in some embodiments,covalent binding of an affinity tag to an appropriately functionalizedsurface. For example, the affinity tag can be part of the proteinencoded by a DNA and can be captured to the surface of an array bycovalent attachment to an appropriate chemical compound that recognizesthe tag.

The contents of all references cited herein (inclusive of patents,patent applications, and patent application publications) are herebyincorporated by reference.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing examples of homo-functional surface bindingagents.

FIG. 2 is a diagram showing examples of hetero-functional surfacebinding agents.

FIG. 3 is a drawing showing an example of a cross-linker used in thearrays.

FIG. 4 is a diagram showing use of sulfate-dextran as a poly-blocker ofamino silane substrate.

DETAILED DESCRIPTION

Protein arrays can be constructed by immobilizing nucleic acids thatencode target proteins onto a surface of a substrate. A translationeffector is contacted to the substrate so that the nucleic acids areexpressed, producing proteins that are then immobilized in situ orotherwise stably attached. Such a protein array is referred to as anucleic acid programmable protein arrays (NAPPA). The proteins aretypically expressed with a tag that can be used to capture the protein,e.g., to the array surface, or to detect it.

As further described herein, array surfaces can include variousmodifications, for example, to facilitate the attachment of nucleicacids and proteins. Nucleic acids can be transiently attached, e.g.,until after translation, and then removed. In addition, array surfacescan be modified, e.g., with a poly-blocker, to reduce non-specificbinding and other interactions with the array surface.

Homo-Functional Binding Surfaces

In one aspect, the disclosure features nucleic acid and protein arrayswith surfaces modified by homo-functional surface chemistry(homo-functional binding groups) (see, e.g., FIG. 1). For example,surfaces can be activated with homo-functional groups (e.g., primaryamino groups, aldehyde groups, epoxy groups, carboxyl groups) and can beused immobilize biomolecules.

Aldehyde- and epoxy-groups are stable at neutral pH values, even in wetconditions, allowing for long-term storage and/or transport beforeimmobilization of biomolecules. In addition, substrates activated withepoxy- or aldehyde-groups can react with various common nucleophilicgroups on the protein surfaces. For example, epoxy groups can react withamino, hydroxyl or thiol moieties; aldehyde groups can react with aminomoieties. Such interactions can produce strong and very stable linkages(e.g., secondary amino bonds, ether bonds, thioether bonds) with minimalchemical modification of the biomolecules because, for example, pKvalues of the new secondary amino groups are very similar to those ofthe pre-existing primary ones. The epoxy groups can be used to formcovalent linkage to proteins, e.g., streptavidin, antibodies, and otheruseful proteins, particularly binding proteins that can be attached to aNAPPA array prior to translation.

Temporary Nucleic Acid Disposal

In some implementations, it is useful to dispose nucleic acids onlytemporarily on an array surface. The nucleic acids can be used for adesired purpose and then removed (e.g., washed away or digested). Forexample, the nucleic acids can be transcribed and/or translated and thenthe surfaces are treated to remove the nucleic acid, e.g., DNA and RNA.

The nucleic acids can be removed by a variety of methods. Preferably,the conditions for removing the nucleic acid do not affect proteinswhich are immobilized to the array surface after translation.

For example, the array surface can include surface features that bind tonucleic acids under limited conditions. The conditions can be altered toremove the nucleic acids when desired. Preferably, the conditions forremoving the nucleic acid do not affect protein attachment to the arraysurface. In one embodiment, the surface can includemono-amino-N-aminoethyl (MANAE) which binds to nucleic acid.

In one embodiment, the substrate includes ethylenediamine or a compoundthat has the formula NH₂—[CH2]-NH₃ ⁺. n is typically greater than 1,e.g., between 2 and 8, e.g., 2, 3, 4, or 5. The substrate can bind thenucleic acid using a pH-sensitive interaction. The step of washing offthe nucleic acid can include altering the pH. The use of such aliphaticdiamines allows for easy adsorption and/or desorption of DNA due todifferent pK values of the two amino groups (primary and secondary). Thepresence of two amino groups in a short aliphatic chain (2-6 carbons)results in decreased pK of the primary amino group. For example, at a pHbetween 6 and 6.5, a diamine has a double positive charge, because bothprimary and secondary amino groups are protonated. However, at pH 8.5,only the secondary amino group is protonated, allowing for desorption ofpreviously adsorbed DNA. Thus, nucleic acid, absorbed to diamine atabout pH 6-6.5 can be removed by increasing pH conditions to about 8.5or greater. Changing the buffering agent in a solution is one method foraltering the pH.

The array can also be treated with an agent that modifies, e.g.,digests, nucleic acids, but which does not substantially affect protein.In one embodiment, the surface can be treated with DNase to remove thebound DNA. The surface can also be treated with RNase, e.g., to removetranscripts, tRNAs, and ribosomes.

The disclosure also features primary amino group substrate for nucleicacid binding, e.g., DNA binding. In one embodiment, high concentrationof salt (e.g., 3-4M NaCl) can be used to wash away the nucleic acid.

Fc Domain Binding Agents

A substrate surface can also be modified to include a non-protein agent(e.g., a chemical moiety) that binds to the Fc region of an antibody.For example, the surface can include a boronate group, or a metalchelating group, e.g., immobilized iminodiacetic acid (IDA) able tochelate metals. The metal chelating group can be used immobilize copper,zinc, nickel, or other metal (e.g., any divalent metal). The surface canbe used to attach a protein that includes an Fc region of an antibody ora fragment of the Fc region that interaction with such agents.

The surface can be used to attach antibodies or Fc region-containingproteins that bind to proteins that are subsequently translated on thearray or can be used to attach translated proteins themselves. Forexample, the surface can include an antibody that recognizes an epitopetag and the nucleic acids disposed on the array surface includesequences that encode target proteins in frame with the epitope tag. Inanother example, the translated proteins themselves can include a region(such as an Fc region) which interacts with the surface agents, e.g.,the boronate group or the metal chelating group. Such a region can be afolded protein domain, e.g., a sequence of at least 30 amino acids. Forinstance the region does not include a poly-histidine tract.

Surfaces with Hetero-Functional Groups

The substrate surface can include hetero-functional (or multifunctional)groups to provide different functions to a single surface. For example,the different groups can be used to covalently bind and immobilizedifferent biomolecules. The different groups can be used to bind andabsorb and then covalently immobilize a compound.

For example, the multifunctional supports can include: (a) one or morefunctional groups that absorb proteins by non-covalent interaction(e.g., by ionic exchange (e.g., amino groups), by phenyl-boronicadsorption, or by IMAC adsorption) and (b) one or more functional groupsthat covalently immobilize the biomolecule (e.g., epoxy groups). FIG. 2provides examples of hetero-functional binding agents.

Proteins can be disposed on a surface containing a heterofunctionalgroup and maintained under conditions that permit generation of covalentlinkages between nucleophilic groups of proteins (primary amino, thiol,and hydroxyl) and a layer of epoxy groups on the surface.

Another exemplary surface includes diamine and carboxyl groups. Thediamine can bind a nucleic acid, e.g., DNA, and the carboxyl canimmobilize an antibody that can capture a protein expressed from thenucleic acid.

The disclosure also features other useful combinations ofhetero-functional groups, e.g., epoxy-amino, amino-phenyl-boronic,amino-IMAC and others. For example, one combination can be used to bindDNA and protein (i.e., DNA-binding protein, such as avidin orstreptavidin that can bind to biotinylated DNA), e.g., an amine, e.g., adiamine, can preferentially bind DNA, while an epoxy group can bind aprotein (with some low cross-reactivity). Another combination can beused to recruit capture antibody and DNA carrier protein and thencovalently attach the protein, e.g., an amine can recruit the protein bycharge close to an epoxy group to facilitate covalent binding. Inanother combination, a protein can be bound with an amine or epoxy and acapture antibody can be oriented with IMAC or boronate.

See generally, e.g., Mateo et al., 2003 Biomacromolecules 4:772-777;Mateo et al., 2003 Biotechnology Progress 19: 1056-1060.

Cross-Linkers

The disclosure also features bi-functional and poly-functionalcross-linkers to stabilize the array reagents and increase bindingcapacity. Cross-linkers can increase binding capacity by allowingbinding of increased number of groups and providing spacer arms.Poly-functional cross-linkers can provide stoichiometry to bindingagents.

In one embodiment, the bi-functional cross-linker is a glutaraldehydethat binds primary amino groups. In another embodiment, thebi-functional cross-linker isN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide HCl that can bind primaryamino groups and carboxyl groups (EDCl).

In one embodiment, the poly-functional cross-linker is analdehyde-dextran that can bind primary amino groups and can be used tocreate aggregates of homo-functional and hetero-functional groups (seeFIG. 3). Aldehyde-dextran can also be used to bind detection antibodiescoupled to fluorophore (or to another detection molecule) to increasesignal.

Substrates

Materials.

Both solid and porous substrates are suitable for recipients for thenucleic acids and proteins described herein. In one embodiment, thesubstrate is a solid substrate. Potentially useful solid substratesinclude: glass (e.g., functionalized glass, a glass slide, poroussilicate glass, a single crystal silicon, quartz, UV-transparent quartzglass), plastics and polymers (e.g., polystyrene, polypropylene,polyvinylidene difluoride, poly-tetrafluoroethylene, polycarbonate,PDMS, acrylic), metal coated substrates (e.g., gold), siliconsubstrates, latex, and membranes (e.g., nitrocellulose, nylon). Thesolid substrate can have a surface suitable for mass spectroscopy (e.g.,it is a mass spectroscopy plates for MALDI), for surface plasmonresonance (SPR), or other analytical technique.

In another embodiment, the substrate is porous, e.g., a gel or matrix.Potentially useful porous substrates include: agarose gels, acrylamidegels, sintered glass, dextran, meshed polymers (e.g., macroporouscrosslinked dextran, sephacryl, and sepharose), and so forth.

The substrate can include beads, e.g., beads disposed on a generallyplanar surface or beads in solution.

Substrate Properties.

The substrate can be opaque, translucent, or transparent. The addressescan be distributed, on the substrate in one dimension, e.g., a lineararray; in two dimensions, e.g., a planar array; or in three dimensions,e.g., a three dimensional array. The solid substrate may be of anyconvenient shape or form, e.g., square, rectangular, ovoid, or circular.In another embodiment, the solid substrate can be disc shaped andattached to a means of rotation.

In one embodiment, the substrate contains multiple addresses(positionally distinguishable locations), e.g., at least 1, 10, 100,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ or more addresses per cm². Thecenter to center distance can be 5 mm, 1 mm, 100 μm, 10 μm, 1 μm, 100 nmor less. The longest diameter of each address can be 5 mm, 1 mm, 100 μm,10 μm, 1 μm, 100 nm or less. In one embodiment, each addresses contains0 μg, 1 μg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 0.1 pg, or less ofthe nucleic acid or protein. In another embodiment, each addresscontains 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ or more molecules ofthe nucleic acid or protein.

The substrate can include a coated surface, e.g., a metal coated surfacesuch as a gold surface, titanium, or chromium surface. The surface canhave a contact angle of between 20-70° or between 33-50° or 50-70°,e.g., about 64°. The surface may include a polymer coat (e.g., on glassor on the metal coat). The polymer can include, e.g., a reactive end,e.g., for attachment to a protein or to an anchoring agent. Exemplarytermini for polymers include amines and activated esters. Exemplarypolymers include alkyl chains and polyethylene glycol, and polymers thatinclude a region, e.g., a hydrophobic and hydrophilic region, e.g., analkyl region and a polyethylene glycol region. The substrate can includediscrete regions of reactivity, e.g., a set of selective regions thatinclude polymers with a reactive end. The regions of reactivity can be,for example, regularly spaced from one another.

Substrate Modification.

The substrate can be modified to facilitate the stable attachment oflinkers, capture probes, or binding agents, e.g., as described herein.Generally, a skilled artisan can use routine methods to modify asubstrate in accordance with the desired application. The following arenon-limiting examples of substrate modifications.

A surface can be amidated, e.g., by silylating the substrate, e.g., withtrialkoxyaminosilane. Silane-treated surface can also be derivatizedwith homobifunctional and heterobifunctional linkers. The substrate canbe derivatized, e.g., so it has a hydroxy, an amino (e.g., alkylamine),carboxyl group, N-hydroxy-succinimidyl ester, photoactivatable group,sulfhydryl, ketone, or other functional group available for reaction.The substrates can be derivatized with a mask in order to onlyderivatized limited areas; a chemical etch or UV light can be used toremove derivatization from selected regions.

Thus, for the preparation of glass slides, options are to derivatize theindividual spots, or to derivatize the entire slide then use a physicalmask, chemical etch, or UV light to cover or remove the derivatizationin the areas between spots.

Partitioned Substrates.

In one preferred embodiment, each address is partitioned from all otheraddresses in order to prevent unique molecules from diffusing to otheraddresses. The following are possible marcomolecules which must remainlocalized at the address: a template nucleic acid encoding the testamino acid sequence; amplified nucleic acid encoding the test amino acidsequence; mRNA encoding the test amino acid sequence; ribosomes, e.g.,monosomes and polysomes, translating the mRNA; and the translatedpolypeptide. Other macromolecules that must remain localized at theaddress include peptides, polypeptides, and proteins.

The substrate can be partitioned, e.g., with depressions, grooves,photoresist. For example, the substrate can be a microchip withmicrochannels and reservoirs etched therein, e.g., by photolithography.Other non-limiting examples of substrates include multi-welled plates,e.g., 96-, 384-, 1536-, 6144-well plates, and PDMS plates. Suchhigh-density plates are commercially available, often with specificsurface treatments. Depending on the optimal volume required for eachapplication, an appropriate density plate is selected. In anotherembodiment, the partitions are generated by a hydrophobic substance,e.g., a Teflon mask, grease, or a marking pen (e.g., Snowman, Japan).

In one embodiment, the substrate is designed with reservoirs isolated byprotected regions, e.g., a layer of photoresist. For example, for eachaddress, a translation effector can be isolated in one reservoir, andthe nucleic acid encoding a test amino acids sequence can be isolated inanother reservoir. A mask can be focused or placed on the substrate, anda photoresist barrier separating the two reservoirs can be removed byillumination. The translation effector and the nucleic acid reservoirsare mixed. The method can also include moving the substrate in order tofacilitate mixing. After sufficient incubation for translation to occur,and for the nascent polypeptides to bind to a binding agent, e.g., anagent attached to the substrate, additional photoresist barriers can beremoved with a second mask to facilitate washing a subset or all theaddresses of the substrate, or applying a second compound to eachaddress.

Planar Substrates.

In another embodiment, the addresses are not physically partitioned, butdiffusion is limited on the planar substrate, e.g., by increasing theviscosity of the solution, by providing a matrix with small pore sizewhich excludes large macromolecules, and/or by tethering at least one ofthe aforementioned macromolecules. In some cases, the addresses aresufficiently separated that diffusion during the time required fortranslation does not result in excessive displacement of the translatedpolypeptide to an address other than its original address on the array.In yet another embodiment, modest or even substantial diffusion toneighboring addresses is permitted. Results, e.g., a signal of a label,are processed, e.g., using a computer system, in order to determine theposition of the center of the signal. Thus, by compensating for radialdiffusion, the unique address of the translated polypeptide can beaccurately determined.

Non-planar substrates can also be used. For example, the non-planarsubstrates may include invaginations or pores.

Three-Dimensional Substrates.

A three-dimensional substrate can be generated, e.g., by successivelyapplying layers of a gel matrix on a substrate. Each layer can contain aplurality of addresses. The porosity of the layers can vary, e.g., sothat alternating layers have reduced porosity.

In another embodiment, a three-dimensional substrate includes stackedtwo-dimensional substrates, e.g., in a tower format. Eachtwo-dimensional substrate is accessible to a dispenser and detector.

A three-dimensional substrate can include beads, e.g., beads positionedon a generally planar substrate, or beads in solution.

Gold and Other Metallic Surfaces

In some cases, it is useful to provide an array of proteins on aconductive surface, e.g., metallic surface, e.g., a gold or gold-coatedsurface, silver or silver-coated surface, titanium or titanium-coatedsurface, chromium or chromium-coated surface, and/or indium oxide orindium-oxide-coated surface. Gold-coated surfaces can be derivatized tobind proteins. In one embodiment, thiol-terminating alkanes can be usedto form self-assembling monolayers on gold. Alkane linkers can bedesigned to contain varying lengths of carbon chains (i.e., 3 to 20) anda variety of terminating chemical moieties, e.g., amines, hydroxyls,aldehydes, nickel, activated esters and/or ethylene glycols. In oneembodiment, to derivatize the surface, the linkers can be prepared invarying concentrations (e.g., 0.001 mM to 100 mM) and incubated with thesurface (e.g., for 1 to 24 hours). One method of preparing proteins(thiols) on a gold surface is provided in an Example below.

To improve adherence of the proteins to the surface, it can be useful toattach aggregates of proteins to the surface rather than individualmolecules to the surface, e.g., the metallic surface.

Aggregates can be formed, e.g., by crosslinking, e.g., using ahomo-bifunctional or hetero-bifunctional crosslinker. Examples of suchhetero-bifunctional crosslinkers include: amine-carboxyl reactivecrosslinkers, amine-sulfhydryl reactive crosslinkers,sulfhydryl-hydroxyl reactive (e.g., N-[p-Maleimidophenyl]isocyanate).Examples of homo-bifunctional crosslinkers include amine or sulfydrylreactive moieties, e.g., activated esters, dextran, imidoesters,sulfosuccinimidyl suberate, and malemide. Specific examples of aminereactive cross linkers include Dimethyl adipimidate.2HCl and Dimethyl3,3″-dithiobispropionimidate.2 HC, and specific examples of sulfhydrylreactive crosslinkers include Bis-Maleimidoethane, Sulfosuccinimidyl6{3′(2-pyridyldithio)-propionamido]hexanoate (SPDP), Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), andNHS-PEO₂₋₁₂-maleimide(succinimidyl-[N,N-maleimidopropionamido)-diethylelenglycol]ester.

The aggregates can include greater than 10², 10³, 10⁴, or 10⁵ molecules(e.g., identical molecules or a mixture of different molecules). Theaggregate can be at least 0.001, 0.01, or 0.1 nm in diameter, e.g., lessthan 0.2, 0.1, or 0.02 nm in diameter.

Transcription Effectors for NAP PA Applications

RNA-directed RNA polymerases and DNA-directed RNA polymerases are bothsuitable exemplary transcription effectors.

DNA-directed RNA polymerases include bacteriophage T7 polymerase, phageT3, phage φII, Salmonella phage SP6, or Pseudomonas phage gh-I, as wellas archeal RNA polymerases, bacterial RNA polymerase complexes, andeukaryotic RNA polymerase complexes.

T7 polymerase is a preferred polymerase. It recognizes a specificsequence, the T7 promoter (see e.g., U.S. Pat. No. 4,952,496), which canbe appropriately positioned upstream of an encoding nucleic acidsequence. Although, a DNA duplex is required for recruitment andinitiation of T7 polymerase, the remainder of the template can be singlestranded. In embodiments utilizing other RNA polymerases, appropriatepromoters and initiations sites are selected according to thespecificity of the polymerase.

RNA-directed RNA polymerases can include Qβ replicase, and RNA-dependentRNA polymerase.

Translation Effectors for NAPPA Applications

In one embodiment, the transcription/translation mix is in a minimalvolume, and this volume is optimized for each application. The volume oftranslation effector at each address can be less than about 10⁻⁴, 10⁻⁵,10⁻⁶, 10⁻⁷, 10⁻⁵, or 10⁻⁹ L. During dispensing and incubation, the arraycan be maintained in an environment to prevent evaporation, e.g., bycovering the wells or by maintaining a humid atmosphere.

In another embodiment, the entire substrate can be coated or immersed inthe translation effector. One possible translation effector is atranslation extract prepared from cells. The translation extract can beprepared e.g., from a variety of cells, e.g., yeast, bacteria, mammaliancells (e.g., rabbit reticulocytes), plant cells (e.g., wheat germ), andarchaebacteria. In a preferred embodiment, the translation extract is awheat germ agglutinin extract or a rabbit reticulocyte lysate. Inanother preferred embodiment, the translation extract also includes atranscription system, e.g., a eukaryotic, prokaryotic, or viral RNApolymerase, e.g., T7 RNA polymerase. In a preferred embodiment, thetranslation extract is disposed on the substrate such that it can beremoved by simple washing. The translation extract can be supplemented,e.g., with additional amino acids, tRNAs, tRNA synthases, and energyregenerating systems. In one embodiment, the translation extract alsoinclude an amber, ochre, or opal suppressing tRNA. The tRNA can bemodified to contain an unnatural amino acid. In another embodiment, thetranslation extract further includes a chaperone, e.g., an agent whichunfolds or folds polypeptides, (e.g., a recombinant purified chaperones,e.g., heat shock factors, GroEL/ES and related chaperones, and so forth.In another embodiment, the translation extract includes additives (e.g.,glycerol, polymers, etc.) to alter the viscosity of the extract.

Affinity Tags

An amino acid sequence that encodes a member of a specific binding paircan be used as an affinity tag. The other member of the specific bindingpair is attached to the substrate, either directly or indirectly. US2005/0048580 describes various exemplary affinity tags and bindingagents that can be used to attach a translated protein to the substrate.

Deposit of Nucleic Acid Sequences on Arrays

The substrate and the liquid-handling equipment are selected withconsideration for required liquid volume, positional accuracy,evaporation, and cross-contamination. The density of spots can depend onthe liquid volume required for a particular application, and on thesubstrate, e.g., how much a liquid drop spreads on the substrate due tosurface tension, and the positional accuracy of the dispensingequipment.

Numerous methods are available for dispensing small volumes of liquidonto substrates. For example, U.S. Pat. No. 6,112,605 describes a devicefor dispensing small volumes of liquid. U.S. Pat. No. 6,110,426describes a capillary action-based method of dispensing known volumes ofa sample onto an array. The dispensed material can include a mixturedescribed herein, e.g., a nucleic acid and a binding agent, or a nucleicacid physically associated with an attachment moiety and, optionally, abinding agent.

Nucleic acid spotted onto slides can be allowed to dry by evaporation.Dry air can be used to accelerate the process.

A nucleic acid disposed on the array can be amplified directly on thearray, by a variety of methods, e.g., PCR; rolling circle amplification,isothermal RNA amplification or NASBA, and strand displacementamplification.

Nucleic Acid Sequences

Any appropriate nucleic acid, e.g., a nucleic acid for translation in aNAPPA application, can be disposed at an address of the array. Thenucleic acid can be an RNA, single stranded DNA, a double stranded DNA,or combinations thereof. For example, a single-stranded DNA can includea hairpin loop at its 5′ end which anneals to the T7 promoter sequenceto form a duplex in that region. The nucleic acid can be anamplification products, e.g., from PCR (U.S. Pat. Nos. 4,683,196 and4,683,202); rolling circle amplification (“RCA,” U.S. Pat. No.5,714,320), isothermal RNA amplification or NASBA (U.S. Pat. Nos.5,130,238; 5,409,818; and 5,554,517), and strand displacementamplification (U.S. Pat. No. 5,455,166).

In one embodiment, the nucleic acid, e.g., DNA, can include a specificoverhang sequence that can hybridize to complementary nucleic acid,e.g., DNA overhang sequence of, e.g., a gene sequence to be expressed.In this embodiment, a nucleic acid sequence with a specific overhangsequence is deposited at an address of the array, a mix of nucleic acidsequences that include corresponding overhangs are generated, the mix ofnucleic acid sequences is added to the array, and the complementaryoverhangs of the added nucleic acids hybridize to the appropriateoverhangs of the deposited nucleic acids.

In one embodiment, the sequence of the encoding nucleic acid is knownprior to being disposed at an address. In another embodiment, thesequence of the encoding nucleic acid is unknown prior to disposal at anaddress. For example, the nucleic acid can be randomly obtained from alibrary. The nucleic acid can be sequenced after the address on which itis placed has been identified as encoding a polypeptide of interest.

A variety of schemes for depositing the coding nucleic acids areavailable. Exemplary methods include binding of different forms of nakedDNA (supercoiled, nicked circular, linear) either by direct adsorptionor by UV crosslinking to variously treated surfaces, the binding of DNAmodified by the incorporation of surface reactive nucleotides, and theuse of surface linking agents such as DNA binding proteins and/orhetero-bifunctional intercalating agents. Various exemplary approachesto immobilize nucleic acids can be found in Application US 2005/0260653.

As further described herein, the coding nucleic acid can be temporarilyimmobilized to the substrate. For example, the coding nucleic acids aredeposited on the substrate, translated, and then removed after thetranslated protein is immobilized to the substrate.

Nucleic acids can be attached to the surface by a linker, e.g., apolymeric linker. The linker can vary in length, e.g., a population oflinker molecules is used such that molecules of the nucleic acid at aparticular position are attached by linkers with a variety of lengths,e.g. to generate a three-dimensional surface array. Coupling DNA to alinker can provide a variety of advantages. For example, the linker canincrease ribosome access to the DNA. In some implementations,biotinylation of nucleic acids is used to distance nucleic acids awayfrom the surface, generating a three-dimensional array.

Blocking Agents

Arrays can be used to detect specific events, e.g., specific bindinginteractions between a capture probe and materials in an analyte.Observations of specific events, however, are complicated bynon-specific ones, for example, non-specific binding events.

Arrays can be modified by a polymer that contains multiple functionalgroups in close proximity. The polymer, termed “poly-blocker,” isgenerally a non-proteinaceous polymer, yet has properties similar to alarge protein. The poly-blocker is usually a homopolymer or aheteropolymer synthesized by the random addition of a set of blockco-polymers. Typically the poly-blocker does not include a peptide bond,nor is a nucleic acid. In select cases in which the poly-blocker doesinclude a peptide bond (e.g., poly-lysine or poly-arginine), thepoly-blocker includes two or fewer types of amino acids or anon-programmed sequence of amino acids. Examples of poly-blockersinclude polyanions, such as sulfate-dextran, carboxymethyl-dextran,aspartic-dextran and polycations.

The poly-blocker can be, for example, between 1-10 kDa in molecularweight, e.g., have an average molecular weight of between 1-3, 3-5,5-10, or 8-15 kDa. The poly-blocker can be linear or branched.

The poly-blocker can be attached to the array surface by covalent ornon-covalent interactions. In some cases the surface is charged and thepoly-blocker is chosen to have the opposing charge such thatelectrostatic forces adhere the poly-blocker to the surface.

In one embodiment, positively charged amine-coated surfaces are used andan effective method to block these surfaces can be the use of and otherpoly-blockers with opposite charge to the surface charge. In anotherembodiment, the surface of the array can be activated with carboxylgroups, and the poly-blocker that is applied is a poly-cation, such aspoly-lysine, poly-arginine, DEAE-dextran, and poly-ethylenimine (PEI).

Covalent bond formation between the poly-blocker and the surface can beused to adhere the poly-blocker to the surface. The poly-blocker and thesurface are chosen to have complementary chemistry. For example, thesurface includes aldehyde groups and the poly-blocker has an aminomoiety (e.g., a primary amine group or a secondary amino group). Inanother example, the surface includes activated esters and thepoly-blocker has an amino moiety.

Poly-blockers can be added at any stage of array preparation. Forexample, in the case of a NAPPA array, the poly-blocker can be used atone or more of the following stages:

1) before transcription and translation (pre-blocking)

2) during transcription and/or translation (e.g., to a translationextract such as a reticulocyte lysate);

3) after transcription and/or translation;

4) before or concurrent with detection (e.g., the poly-blocker can beincluded with a detection reagent such as a “query” antibody).

Poly-blockers can be added prior to an analyte, e.g., to block availablenon-specific sites on a surface prior to contacting the analyte thesurface. Poly-blockers can also be used to modulate the stringency ofprotein-interactions. For example, the poly-blocker can be included insolution when a capture probe is contacted with an analyte.

Exemplary Applications

The nucleic acid and protein arrays described herein can be used in anumber of applications. Non-limiting examples are described as follows.The regulation of cellular processes, including control of geneexpression, can be investigated by examining protein-protein,protein-peptide, and protein-nucleic acid interactions; antibodies canbe screened against an array of potential antigens for profilingantibody specificity or to search for common epitopes; proteins can beassayed for discrete biochemical activities; and the disruption ofprotein-ligand interactions by synthetic molecules or the directdetection of protein-synthetic molecule interactions can aid drugdiscovery. Given the versatility of array technologies, elements at eachaddress are easily customized as appropriate for the desiredapplication.

Nucleic acid and protein arrays can be used to characterize biomarkersand autoantibodies. For example, nucleic acids can be bound andexpressed on an array surface and screened with patient serum toidentify novel immunodominant antigens. A patient's immune system canproduce humoral responses to antigens, these antigens may be proteinsthat are normally found in the body but depending on theirpathophysiology there may be alterations in protein expression,mutation, degradation, or localization which may make the proteinimmunogenic. This can be used to evaluate subject having or suspected ofhaving autoimmune diseases. The humoral response can also be proteinsthat are either pathogenic or viral in origin. Therefore by expressingpotential antigens one could screen with patient sera and identifyimmunodominant antigens derived from tumors (breast, colorectal,prostate etc.), autoimmune rheumatic diseases, pathogenic, and/or viral.The identification of immunodominant antigens with high sensitivity andspecificity can be used for early detection of disease, to developvaccines, and monitor disease progression and therapy. For some of theseapplications, the protein can be configured to include evaluatedantigens to be used as a diagnostic tool.

Protein arrays can be used for analysis using label-free systems, suchas mass spectrometry, calorimetry, and/or surface plasmon resonance.Most of these applications are implemented using substrates that havespecific surface chemistry, such as surfaces with properties withsuitable conductivity and ability to generate plasmons. An exemplaryprotein array has been adapted to the gold surface, which satisfies thedemands of these label free detection systems.

The arrays can be probed with complex protein mixtures such as celllysates, tissue, patient sera, etc. In this approach, multiple bindingevents may take place at each feature of the array, resulting in varyingcomposition and amounts of bound material from feature to feature. Usinglabel-free systems, these binding events can be measured and, in somecases, the identity, relative amounts and kinetics of the binding can bedetermined. This information can be used to generate patterns which canthen be used to generate signatures that are specific to the sample. Theability to create unique signatures may help discern the presence ofdisease, biological agents, or changes in biological response.

On the other hand, nucleic acid and proteins arrays can be probed with adefined query, rather than a complex mixture. This avoids the need forlabeling query molecules, such as small molecules, peptides, and nucleicacids, which may affect their binding kinetics. Using this approach, onecan identify both specific and non-specific interactions with proteinson the array. For example, this approach could be applied to determinespecificity of antibodies, small molecules, enzymes and receptors, aswell as any off target interactions. Moreover, fragments of the bindingproteins can be expressed to identify the interacting domains.

A nucleic acid or protein array can be used to detect a specific proteinactivity. Each address of the array is contacted with the reagentsnecessary for an activity assay. Then, an address having the activity isdetected to thereby identify a protein having a desired activity. Anactivity can be detected by assaying for a product produced by a proteinactivity or by assaying for a substrate consumed by a protein activity.

Protein Interaction Detection

Nucleic acid and protein arrays, e.g., NAPPA arrays, can be used todetect protein-protein interactions. Moreover, the arrays can be used togenerate a complete matrix of protein-protein interactions such as for aprotein-interaction map (see, e.g., Walhout et al., Science 287:116-122, 2000; Uetz et al., Nature 403, 623-631, 2000); and Schwikowski(2000) Nature Biotech. 18:1257). The matrix can be generated for thecomplete complement of a genome, proteins known or suspected to beco-regulated, proteins known or suspected to be in a regulatory network,and so forth.

In some NAPPA applications, the detection of protein-proteininteractions, e.g., between a first and a second protein, entailsproviding at an address a nucleic acid encoding the first polypeptideand an affinity tag, and a nucleic acid encoding a second polypeptideand a recognition tag, e.g., a recognition tag described below. In oneembodiment, the detection of protein-protein interactions, e.g., betweena first and a second protein, entails providing at an address a nucleicacid encoding the first polypeptide and an affinity tag, and a nucleicacid encoding a second polypeptide, where the second polypeptide isdetected with the use of, e.g., an antibody or a reagent specific forthe second peptide.

In one NAPPA embodiment, after translation of both nucleic acids, thearray is washed to remove unbound proteins and the translation effector.Detection of an address at which the second polypeptide remains bound isindicative of a protein-protein interaction between the first and secondpolypeptide of that address.

In another NAPPA embodiment, a third or competing polypeptide can bepresent during the binding step, e.g., a third encoding nucleic acidsequence lacking a tag can be included at the address.

In another embodiment, a third peptide that binds to or bridges thefirst and the second polypeptide can be added. The third peptide canenable the interaction of the first and the second peptides, i.e., thefirst and the second peptides do not interact in the absence of thethird peptide.

In yet another embodiment, the stringency or conditions of the bindingor washing steps are varied as appropriate to identify interactions atany range of affinity and/or specificity.

Protein-Small Molecule Screens

The arrays described herein can be used to identify a polypeptide thatbinds a small molecule. The small molecule can be labeled, e.g., with afluorescent probe, and contacted to a plurality of addresses on thearray (e.g., prior, during, or after translation of the programmingnucleic acids). The array can be washed after maintaining the array suchthat the small molecule can bind to a polypeptide with an affinity tag.The signal at each address of the array can be detected to identify oneor more addresses having a polypeptide that binds the small molecule.

Other signal detection methods include surface plasmon resonance (SPR)fluorescence polarization (FP), infrared spectroscopy, andnano-gravimetry. Methods for using FP are described, for example, inU.S. Pat. No. 5,800,989. Methods for using SPR are described, forexample, in U.S. Pat. No. 5,641,640; and Raether (1988) Surface PlasmonsSpringer Verlag.

In another embodiment, the invention features a method of identifying asmall molecule that disrupts a protein-protein interaction. The array,e.g., a NAPPA array, is programmed with a first and a second nucleicacid which respectively encode a first and second polypeptide whichinteract. The first polypeptide includes an affinity tag and secondpolypeptide includes a recognition tag. A unique small molecule iscontacted to an address of the array (e.g., prior, during, or aftertranslation of the programming nucleic acids). The array can be washedafter maintaining the array such that the small molecule, the first andthe second polypeptide can interact. The signal at each address of thearray is detected to identify one or more addresses having a smallmolecule that disrupts the protein-protein interaction.

An exemplary application that exploits the ability to screen for smallmolecule interactions with proteins is the pre-clinical evaluation of alead drug candidate. Drug toxicities often result not from the intendedactivity on the target protein, but some activity on an unrelatedbinding protein(s). Even when these adventitious binding proteins do notcause toxicity, they can adversely affect the drug's pharmacokinetics. Aprotein array can be used to identify these adventitious binders. Forexample, the drug candidate is contacted to a protein array thatincludes various potential targets, e.g., cellular proteins.

The small molecule screen could become a rapid and powerful platform bywhich medicinal chemistry and SAR could be performed. Chemicalmodifications of small molecules could be tested against the nucleicacid or protein arrays to see if changes improve specificity. Compoundscould be exposed first to hepatic lysates or other metabolic extractsthat mimic metabolism, in order to create potentially toxic metabolitesthat can also be screened for secondary targets. Recursion of thisprocess could lead to improved specificity and tighter bindingmolecules.

Mass Spectroscopy

The nucleic acid and protein arrays can be used in conjunction with massspectroscopy, e.g., to detect a modified region of the protein. An arrayis prepared as described herein with due consideration for the flatness,conductivity, registration and alignment, and spot density appropriatefor mass spectroscopy.

In one embodiment, the method identifies a polypeptide substrate for amodifying enzyme. In a NAPPA application, each address is provided witha nucleic acid encoding a unique test polypeptide. Each address of thearray is contacted with the modifying enzyme, e.g., a kinase, amethylase, a protease and so forth. The enzyme can be synthesized at theaddress, e.g., by including a nucleic acid encoding it at the addresswith the nucleic acid encoding the test sequence. After sufficientincubation to assay the modification step, each address is proteolyzed,e.g., trypsinized. The resulting peptide mixtures can be subject toMALDI-TOF mass spectroscopy analysis. The combination of peptidefragments observed at each address can be compared with the fragmentsexpected for an unmodified protein based on the sequence of nucleic aciddeposited at the same address. The use of computer programs (e.g., PAWS)to predict trypsin fragments is routine in the art. Thus, each addressof the array can be analyzed by MALDI. Addresses containing modifiedpeptide fragment relative to a predicted pattern or relative to acontrol array can be identified as containing potential substrates ofthe modifying enzyme.

The amount of modifying enzyme contacted to an address can be varied,e.g., from array to array, or from address to address.

For example, this approach can be used to identify phosphorylation bycomparing the masses of peptide fragments from an address having akinase, and an address lacking the kinase. Pandey and Mann (2000) Nature405:837 describe methods of using mass spectroscopy to identify proteinmodification sites.

In another embodiment, in a NAPPA application, the modifying enzyme isvaried at each address, and the test polypeptide, the polypeptide withthe affinity tag for attachment to the substrate, is the same at eachaddress. Both the modifying enzyme and the test polypeptide can besynthesized on the array by translation of encoding nucleic acidsequences. Mass spectroscopy is used to identify an address having amodifying enzyme with specificity for the test polypeptide asenzyme-substrate.

Mass spectroscopy can also be used to detect the binding of a secondpolypeptide to the target protein. In a NAPPA application, a firstnucleic acid encoding a unique target amino acid sequences and anaffinity tag is disposed at each address in the array. A pool of nucleicacids encoding candidate amino acid sequence is also disposed at eachaddress of the array. Each address of the array is translated and washedto remove unbound proteins. The proteins that remain bound at eachaddress, presumably by direct interaction with the target proteins, canthen be detected and identified by mass spectroscopy.

Mass spectroscopy can be used in protein-small molecule screens, e.g.,to detect binding of a test compound, e.g., a small molecule, to thetarget protein. In this embodiment, a first nucleic acid encoding aunique target amino acid sequences and an affinity tag is disposed ateach address in the array. A pool of test compounds, e.g., smallmolecules, is also disposed at each address of the array. Such testcompounds may or may not have to be chemically modified. Each address ofthe array is translated and washed to remove unbound test compounds. Thetest compounds that remain bound at each address, presumably by directinteraction with the target proteins, can then be detected andidentified by mass spectroscopy. Small molecules are well-suited toidentification by mass spectroscopy because, e.g., they fly easily inthe instrument, do not require prior digestion, and can have uniquesignatures.

Transmembrane Proteins

Transmembrane proteins that include one or more transmembrane domainscan be provided on a substrate, e.g., in an array format. Thetransmembrane protein is synthesized in close proximity to a substratethat includes a binding agent that immobilizes the transmembraneprotein. Without being bound by theory, it is postulated thataggregation between transmembrane proteins is avoided, because thetransmembrane proteins are immobilized either during translation orrapidly thereafter, e.g., before aggregation occurs.

In some exemplary implementations, a nucleic acid encoding thetransmembrane protein, including at least one, two, or threetransmembrane domains is deposited on the substrate. In some cases, thetransmembrane protein includes four, five, six, or seven transmembranedomains, e.g., between six and twelve transmembrane domains. The nucleicacid optionally includes a sequence encoding a tag, e.g., a tag forwhich there is a binding agent on the substrate. The sequence encodingthe tag is in frame with the sequence encoding the transmembraneprotein, such that translation of the nucleic acid produces thetransmembrane protein including the tag.

The substrate also includes the binding agent. Generally, the nucleicacids and the binding agent are both located in close proximity on thesubstrate. For example, the nucleic acid encoding the transmembraneprotein can be combined with the binding agent, and the combination canbe disposed on the substrate.

The substrate is then contacted with a translation effector, e.g., atranslation extract such as a reticulocyte lysate. In cases where thedeposited nucleic acid is DNA, the substrate can also be contacted witha transcription effector (e.g., T7 RNA polymerase). In many embodiments,the translation extract does not include lipids, micelles, or bilayerforming detergents or membranes. As a result, the transmembrane proteinsare captured after translation but not inserted into membrane bilayers.

The transmembrane protein can include a full length natural protein, anatural protein without a signal sequence, or a fragment of a naturalprotein, e.g., a fragment that includes at least one transmembranedomain.

In one embodiment the protein includes at least five, six, or seventransmembrane domains. For example, the protein includes exactly seventransmembrane domains. The protein can be a GPCR (G-protein coupledreceptor). The substrate can further include one or more nucleic acidsthat include regions that encode a GPCR binding protein, e.g., a Gprotein subunit, e.g., the α, β, and γ subunits.

The method can further include contacting the substrate with a testcompound, e.g., a candidate ligand or candidate drug compound. Thesubstrate can be evaluated to determine the interaction of the testcompound with the transmembrane protein synthesized at differentlocations on the substrate. For example, the substrate can be evaluatedto determine whether the test compound is bound to the substrate byinteraction with the transmembrane protein. In another example,interaction of the test compound with the transmembrane protein isevaluated by determining whether the conformation of the transmembraneprotein is altered, e.g., using a conformation specific antibody or aspectroscopic probe.

Extracellular Proteins

In one embodiment, an extracellular polypeptide or an extracellulardomain can be displayed on a nucleic acid or a protein array, e.g., aNAPPA array, e.g., by contacting the array with conditions similar tothe extracellular, endoplasmic reticulum, or Golgi milieu. For example,the conditions can be oxidizing or can have a redox potential that isoptimized for extracellular protein production. The array can beadditionally contacted with modifying enzymes found in the secretorypathway, e.g., glycosylases, proteases, and the like.

In another embodiment, in a NAPPA application, the translation effectoris applied in conjunction with vesicles, e.g., endoplasmic reticularstructures, and microsomes. The vesicles can include an affinity tag toanchor the vesicle to the array. In such an embodiment, the encodingnucleic acid need not contain an affinity tag.

An array of extracellular proteins or extracellular protein domains canbe used to identify interactions with other extracellular proteins; oralteration of living cells (e.g., the adhesive properties, motility, orthe secretory repertoire of a cell contacting the extracellularprotein).

Cell-Based Assays

In another embodiment, at least one address of the array, e.g., a NAPPAafter translation of encoding amino acids, is contacted with a livingcell. After contacting the array, the cell or a cell parameter ismonitored. For example, polypeptide growth factors can be arrayed atdifferent addresses, and cells assayed after contact to each address.The cells can be assayed for a change in cell division, apoptosis, geneexpression (e.g., by gene expression profiling), morphology changes,differentiation, proteomics analysis (e.g., by 2-D gel electrophoresisand mass spectroscopy), and specific enzymatic activities.

In one embodiment, a test protein of the array can be detached from thesubstrate of the array, e.g., by proteolytic cleavage at a specificprotease site located between the test sequence and the tag. A releasedtest protein can be internalized or otherwise acted upon by a cell thatis contacted to the array.

In another embodiment, in a NAPPA application, the test polypeptide doesnot have an affinity tag, but is maintained at an address by physicalseparation from other addresses of the plurality. The translationeffector is optionally not washed from the address. Cells are assayedafter being maintained at the address as described above.

A substrate containing a plurality of different proteins can be used toevaluate cellular responses. The substrate can be contacted with cells,e.g., mammalian cells. A response of the cells can be evaluated, e.g.,by imaging the substrate. For example, the cells may include aimage-detectable reagent, e.g., a sensor dye (such as a calciumsensitive dye) or a reporter gene that produces a reporter proteins(e.g., a reporter protein detectable by visual imaging, e.g., luciferaseor a fluorescent protein (e.g., GFP)). Generally the cells are livecells, but fixed or other non-living cells can also be used, e.g., toevaluate binding interactions.

In some embodiments, the substrate further includes a reagent forevaluating the cells. The reagent can be covalently or non-covalentlybound to the substrate. For example, the reagent can be a molecule thatbinds to or detects a cellular component, e.g., a cell-surface proteinor a protein secreted by the cells. Examples of secreted proteinsinclude insulin, growth factors, peptide hormones, and so forth. Thereagent can be an antibody or other binding protein that binds to thecell-surface protein or the secreted protein. The substrate can becontacted with a compound. After, one or more time intervals, cells onthe substrate can be evaluated. For example, the amount of the cellularcomponent that is detected can be evaluated.

As an illustration, the substrate can be contacted with one or morecompounds and evaluated to find compounds that increase or decreasesecretion of a protein.

Different proteins can be evaluated a different addresses on thesubstrate. For example, the reagent can differ among addresses of thesubstrate. The different proteins can be deposited at the differentaddresses or can be deposited in situ.

The substrate can be an array, e.g., an array of proteins prepared by amethod described herein. For example, the array can include nucleicacids, each encoding a polypeptide that includes a tag, and the arraycan further include a binding agent for binding to the tag, such thatexpressed polypeptides are anchored to the substrate by the bindingagent. For example, the expressed polypeptides can be used to detectdifferent compounds at different addresses of the array.

An example of a detection method is an enzyme-linked immunospot(ELISPOT) assay. For example, the assay can detect T-cell responses. Inthis example, immunological cells, e.g., T cells, are exposed to thesubstrate, and the cells are evaluated for a response, e.g., secretionof a particular protein, e.g., an antibody or a cytokine. The cells canbe evaluated for activated T cells release factors, e.g., release ofinterferon gamma (IFN-γ) or interleukin-2 (IL-2).

Accordingly, in one embodiment, a substrate containing a plurality ofdifferent proteins (e.g., antigens, e.g., in full length, fragment, orpeptide form) is provided. The substrate also includes a reagent (e.g.,an antibody) that binds to IFN-γ or IL-2. The substrate is contactedwith T cells in an appropriate medium. At one or more times aftercontact to the substrate, the substrate can be evaluated to determinewhether there is a change in cytokine (e.g., IFN-γ or IL-2) secretion.For example, the substrate can be evaluated using a sandwich assayapproach, e.g., using an antibody. For example, a secondary antibodyspecific for the released protein and linked to a signal molecule canalso be added to visualize areas of captured proteins. The areas ofcaptured proteins can be detected, e.g., as spots of color whenvisualized, around T cells. The assay can be used to detect proteinrelease at the level of single cells.

Other examples of cells that can be contacted to an array include:bacterial cells, yeast cells, plant cells, and animal cells,particularly mammalian cells, e.g., human cells or non-human primatecells. The cells can cells from a hematopoietic lineage, e.g.,hematopoietic stem cells, B cells, and T cells (including CD4+CD8− andCD4−CD8+ T cells and double positive T cells).

Diagnostic Assays

A variety of nucleic acid and protein arrays can be provided fordiagnostic purposes. The array can be used as a screening tool to lookfor antibodies that bind to specific proteins. This could be applied forthe generation of monoclonal antibodies in a high-throughput setting orin the context of measuring immune responses in a patient. ELISAtechniques can be used for detection.

Antigen Arrays.

One class of such arrays is an array of antigens, displayed for thepurpose of determining the specificity of antibodies in a subject. Thearray is set up, or in case of a NAPPA array is programmed, such thateach address represents a different antigen of a pathogen or of a malady(e.g., antigens significant in allergies; transplant rejection andcompatibility testing; and auto-immune disorders).

In one embodiment, the array has antigens from a plurality of bacterialorganisms. Computer programs can be optionally used to predict likelyantigens encoded by the genome of an organism (Pizza et al. (2000)Science 287:1816). In a preferred embodiment, each address has disposedthereon a unique antigen. In another preferred embodiment, each addresshas a plurality of antigens, all being from the same species. Thus, forexample, binding of a subject's antibody to an address indicates thatthe subject has been exposed to a pathogen represented by the address.

In another preferred embodiment, the array is used to track theprogression of complex diseases. For example, diseases with antigenicvariation (e.g., malaria, and trypanosomiasis) can be accuratelydiagnosed and/or monitored by identifying the repertoire of specificantibodies in a subject.

In another embodiment, the array can be used to detect the specifictarget of an autoimmune antibody. For example, isolated antibodies orserum from a subject having type I diabetes are contacted to an arrayhaving islet-cell specific proteins present at different addresses ofthe array.

Antigen arrays also provide a convenient means of monitoringvaccinations and disease exposure, e.g., in epidemiological studies,veterinary quarantine, and public health policy.

Antibody Arrays.

Another class of diagnostic arrays is arrays of antibodies. A variety ofmethods are available for identifying antibodies. Monoclonal antibodiesagainst a variety of antigens are identified. The nucleic acids encodingsuch antibodies are sequenced from the genome of hybridoma cells. Thenucleic acid sequence is used to engineer, e.g., single-chain variantsof the antibody, affibodies, ankyrin repeat proteins, and aptamers. Theencoding nucleic acid sequence can be recombined into an appropriatevector.

In addition, the antibody sequence can be engineered to removedisulfides (Proba K (1998) J. Mol. Biol. 275:245-53). Alternatively,e.g., in NAPPA applications, after translation and washing of the array,the array is subject to oxidizing conditions, e.g., by contacting withglutathione. The antibodies can be coupled to the array withstreptococcal protein G, or S. aureus protein A. Further, specializedantibodies such as modified or CDR-grafted version of naturallyoccurring antibodies devoid of light chains can be used. The antibodiesof camel (e.g., Camelus dromedaries) are naturally devoid of lightchains (Hamers-Casterman C (1993) Nature 363:446-8; Desmyter et al. NatStruct Biol 1996 September; 3(9):803-11).

A patient sample can then be contacted to the array. Non-limitingexamples of patient samples include serum proteins, proteins extractedfrom a biopsy, synovial fluid, cerebrospinal fluid, urine, nippleaspirate, semen, vitreous fluid etc. In addition, cells can be contactedto the array in order to query for antigens displayed on the cellsurface.

In one embodiment, the sample is modified with a compound prior to beingcontacted to the array. For example, the sample can be biotinylated. Inanother embodiment, the sample is unlabelled. Arrays can be designed toidentify proteins associated with various maladies, e.g., to detectantigens associated with cancer at various stages (for example, early,and pre-metastatic stages) or to provide a prediction (for example, toquantitate the abundance of an antigen correlated with a condition).

Proteins can be used as biomarkers. For example, antigens that areassociated with a particular condition can be considered a biomarker.Examples of antigens include CEA, CA-125 and PSA. PSA, for example, canbe used to evaluate risk or presence of prostate cancer.

In addition to detecting protein biomarkers, it is useful to evaluate asubject to detect their antibody or antibody responses. For example, thepresence of an antibody can be an indicator of a disorder, e.g., anautoimmune disorder or a neoplastic disorder.

An alternative format to using an array of capture reagents is to use areverse phase protein blot. Multiple samples can be disposed on anarray. The samples can also include different fractions of an originalsample, e.g., an original sample obtained form a subject. A sample canbe resolved by various methods, e.g., chromatography methods or gelresolution.

Aspects of this application are further illustrated by the followingexamples:

Example Coating Amino Surface with Poly-Anions

In this example, glass slides that have primary amino groups are used.Poly-anions (for example, sulfate-dextran, carboxymethyl-dextran,aspartic-dextrans, glutamic-dextrans etc.) are used as the blockingreagent to reduce non-specific adsorption. FIG. 1 shows blocking withsulfate-dextran. Modification by poly-anions can produce a modifiedsurface that is relatively inert, e.g., with approximately zero netcharge, thereby preventing ionic adsorption.

Dextran-sulfate was prepared by sulphating dextran via clorosulphonicacid-pyridine complex (Nagasawa K. et al., Carbohydrate Research, 1972,21: 420-423; Muhner C. et al., Carbohydrate Research 2001, 331:203-208). Aspartic-dextran was prepared by aldehyde-dextran, previouslygenerated via periodate oxidation (Fuentes M et al., BiotechnologyProgress, 2004, 20, 284-288).

The ionic adsorption of the poly-blocker, just as the ionic adsorptionof proteins, depends on the pH value at which the adsorption processoccurs. In some cases in which the amino-coated surfaces of the arraysare blocked with sulfate-dextran (or another poly-anion), thepoly-blocker treatment can be between pH 7-7.5.

In one embodiment, the array surface is positively charged, andsulfate-dextran is used as the poly-blocker.

The poly-blocker concentration can also be varied. An exemplaryconcentration of sulfate-dextran (in a solution that is applied to thesurface) that effectively reduces non-specific binding to the surface isbetween 0.1 to 10 mg/mL, e.g., about 0.1 to 1 mg/mL or 1 to 5 mg/ml. Forexample, in some embodiments, high concentrations of poly-blocker areavoided as additional poly-blocker could increase surface charge andhence increase ionic adsorption of biomolecules to the surface.

Polymer size can influence the amount of ionic groups per molecule ofthe poly-blocker and can also influence the topography of the surface.Large poly-blockers can cover larger areas but can also generatepoly-blocker brushes, rather than monolayers.

Example 1 Protocol: Array Printing

2 Material/Equipment Plasmid DNA Psoralen-biotin (Pierce 29986) Sodiumacetate (3M, pH 5.5) Isopropanol Ethanol UV Crosslinker, 365 nmwavelength (UVP 95-0228-01) 384 well plate for arraying, Genetix x7020Polyclonal anti-GST antibody (GE Healthcare/Amersham 27457701)Streptavidin (Cortex CE0301B) BS³ Linker (Pierce 21580) Purified GSTprotein (Sigma G5663) Whole mouse IgG antibody (Pierce 31204)Centrifuge, Eppendorf 5810 QArray2 Silica packets (VWR 100489-246)Genetix Bioassay dish dividers (x6026 divider only; x6027 with dish)Corning deep bioassay dish (431111) WellMate Eppendorf Thermomixer

2 Material/Equipment Plasmid DNA Psoralen-biotin (Pierce 29986)

Sodium acetate (3M, pH 5.5)

Isopropanol Ethanol

UV Crosslinker, 365 nm wavelength (UVP 95-0228-01)384 well plate for arraying, Genetix x7020Polyclonal anti-GST antibody (GE Healthcare/Amersham 27457701)

Streptavidin (Cortex CE0301B) BS³ Linker (Pierce 21580)

Purified GST protein (Sigma G5663)Whole mouse IgG antibody (Pierce 31204)

Centrifuge, Eppendorf 5810 QArray2

Silica packets (VWR 100489-246)Genetix Bioassay dish dividers (×6026 divider only; ×6027 with dish)Corning deep bioassay dish (431111)

WellMate Eppendorf Thermomixer

-   -   1) Prepare psoralen-biotin: Stock 10 mg/mL. Working solution 20        ng/μL. Add 10 of the working biotin solution to the 150 μL of        DNA in the Greiner conical plate. A total of 200 ng of biotin        should be added per sample. The Genmate may be used for this        addition on a full plate basis.    -   2) Crosslink for 30 minutes with UV Crosslinker. Use max power        set at 9999. The plates must be uncovered for this step. Total        dose=8800 mJ/cm².    -   3) Add 20 μL of 3 M Sodium Acetate (pH 5.5). Add 120 μL of        isopropanol. These two additions can be done with the Biomek FX.        Cover the plate with an aluminum seal and mix by inverting 3        times.    -   4) Spin at 5300 rcf for 15 mins using the Sorvall RC12        centrifuge.    -   5) Discard the supernatant.    -   6) Add 100 μL of 80% ethanol to each well. The WellMate may be        used for this addition.    -   7) Seal the plate with an aluminum seal and shake at 1000 rpm        for ˜2 mins on the Thermomixer.    -   8) Spin at 5300 rcf for 15 mins using the Sorvall RC12        centrifuge.    -   9) Discard the supernatant.    -   10) Dry the plate, uncovered, in the hood for 10 mins.

Array Sample Preparation:

-   -   1) Prepare master mix. For one 96-well plate prepare        approximately 3 mL of master mix. Master mix contains polyclonal        GST antibody (final: 1:100 dilution or 50 μg/mL), Streptavidin        (final: 3.6 mg/mL) and BS³ linker (final: 1.25 mg/mL or 2 mM).        -   e.g. 3 mL Master Mix:        -   Streptavidin (66 mg/mL stock): 166.5 μL            -   BS³ Linker (50 mg/mL stock): 75.0 μL            -   Polyclonal α-GST (5 mg/mL stock): 30.0 μL            -   AC MQ H₂O: 2728.5 μL    -   2) For robotic master mix addition, aliquot the master mix        evenly into each well of a PCR plate. Each well should contain        enough master mix for the number of plates receiving master mix        (i.e. 20 μL x number of plates) plus a 10 excess. For example,        to add 20 μL master mix to 4 DNA pellet plates, each well of the        master mix PCR plate should contain (4×20 μL)+10 μL=90 μL. This        step is not necessary for manual master mix addition.    -   3) Add 20 μL to each well of the dry DNA pellet plates using the        Biomek FX or a multi-channel pipette. Spin the plates down        briefly and shake the plates rapidly at 1400 rpm for ˜5 mins at        room temperature on the Thermomixer. Briefly spin the plates        down again.    -   4) Transfer 18 μL to 384 array plate. This transfer can be done        using the Biomek FX or a multi-channel pipette.    -   5) Spin the plate down briefly.    -   6) Array, using the appropriate array setup and humidity control        between 60%.    -   7) Put barcode labels on the bottom (non-arrayed) side of each        slide. Maintain the slides order on the deck in numerical order.    -   8) Place the spotted slides, in order, on bioassay dishes with        dividers. Each dish should accommodate approximately 15 slides.        Fill the bottom of each bioassay dish with water so that only        the bottom of the dish is covered. Do not use too much water and        do not spill water onto the arrayed slides. Cover each dish and        carefully stack the dishes together on top of a cart. Cover the        entire stack with a sheet of aluminum foil on top. Wheel the        slides into the cold room for over-night incubation.    -   9) Dry the slides and store at room temperature in a metal rack        in a Lock-n-Lock box with a silica packet.

Protocol: Expression of the NAPPA Slides

Amount Material/Equipment (for 3 slides) HybriWell gaskets (GraceHBW2160-1LA) 3 Cell free expression system i.e. rabbit reticulocytelysate 1 tube (Promega L4610) RNaseOUT (Invitrogen 10777-019)    8 μLDEPC water (Ambion 9906)   160 μL SuperBlock (Pierce 37535)  ~30 mLBlocking solution: 5% Milk in PBS with 0.2% Tween20 ~120 mL PBSProgrammable chilling incubator, with leveling shelves Rocking shakerGenetix Bioassay dish dividers (x6026 divider only; x6027 with dish)Corning deep bioassay dish (431111)

-   -   1 Blocking: ˜1 hr on rocking shaker at room temperature or 4° C.        overnight in the cold room with SuperBlock or milk. Use ˜30 mL        in a pipette box for 4 slides.    -   2 Rinse with Milli-Q water. Dry with filtered compressed air.    -   3 Apply HybriWell gasket to each slide. Use the wooden stick to        rub the areas where the adhesive is to make sure it is well        stuck to the slide all around.    -   4 Pre-heat the incubator to be used for IVT at 30° C.    -   5 Prepare IVT. Each slide will require 130 μL of IVT lysate mix.        Each tube after component addition will contain 400 μL of        lysate mix. Since the lysate tubes cannot be re-frozen, always        try to express slides in batches of some multiple of three.        -   e.g. 1 tube=3 slides=400 μL        -   16 μL TNT buffer        -   8 μL T7 polymerase        -   4 μL of -Met        -   4 μL of -Leu or -Cys        -   8 μL of RNaseOUT        -   160 μL of DEPC water        -   200 μL of reticulocyte lysate    -   6 Add IVT mix from the non-label or non-specimen end. Pipette        the mix in slowly; Gently massage the HybriWell to spread the        IVT mix over the array. Apply the small round port seals to both        ports.    -   7 Place the slides on a bioassay dish with divider on top of the        leveling shelf inside the incubator. Incubate for 1.5 hr at        30° C. for protein expression (e.g., between 29-31° C. or 28-32°        C.), followed by 30 min at 15° C. for the query protein to bind        to the immobilized protein.    -   8 Remove the HybriWell and immerse each slide in milk        immediately; wash with milk three times, 3 minutes each, in a        pipette box. Use about 30 mL milk per wash.    -   9 Block with milk on rocking shaker overnight at 4° C. or at        room temperature for 1 hr.

Protocol: Detection of the NAPPA Arrays

Material/Equipment Amount (for 1 slide) Primary AB, mouse anti-GST (CellSignal 2624) 150 μL of stock solution Primary AB, mouse anti-HA 150 μLof stock solution Secondary AB, HRP-conjugated anti-mouse 150 μL ofstock (Amersham NA931) solution TSA reagent (PerkinElmer SAT704B001EA)150 μL of stock solution Milk (5% Milk in PBS with 0.2% Tween20) 90 mLfor 4 slides at once PBS (pH 7.4) 90 mL for 4 slides at once Coverslips,24 × 60 mm (VWR 48393-106) 3 Lifterslips, 24 × 65 mm 3 (Erie25X65I-2-5251-001-LS) Pipette boxes 1 Scanner, PerkinElmerProScanArray ™

-   -   1) If needed, prepare antibody solutions in SuperBlock: 1:200        mGST, 1:1000 HA, 1:200 anti-mouse, 1:500 anti-human IgG. Store        at 4° C.    -   2) If needed, prepare the TSA stock solution: add ˜1 mL of        Amplification Diluent (from TSA kit) to each of the 5 tubes with        dried TSA. Vortex. Solution will turn bright pink. Pour those 5        solutions back into the diluent bottle. Take another ˜1 mL of        that mix for each tube, vortex again, and add back to the        diluent bottle. Keep this solution at 4° C.    -   3) Apply primary AB (mouse anti-GST or mouse anti-HA) by adding        150 μL to the non-label or non-specimen end of the slide, then        apply a coverslip. Generally, avoid drying the slides during        this step. Incubate for 1 hr at RT; wash with milk on a rocking        shaker (3 times, ˜5 min each). Drain.    -   4) Apply secondary AB (anti-mouse HRP) by adding 150 L to the        non-label or non-specimen end of the slide, place a coverslip.        Again, treat each slide one at a time starting with removal from        the blocking milk through coverslip application. Generally,        avoid drying the slides during this step. Incubate for 1 hr at        RT; wash with PBS (pH 7.4) 3 times, ˜5 min each. Quickly rinse        with Milli-Q water. Drain.    -   5) Apply 150 L TSA mix and place coverslip. Incubate for 10        minutes. Rinse in Milli-Q water; dry with filtered compressed        air.    -   6) Scan.

Example Epoxy-Silane Coating

300 ml of 2% of epoxy silane (3-glycidyloxypropyl)triethoxysilanesolution in acetone was prepared. The slides were treated with epoxysilane coating solution for 15 minutes at room temperature. The slideswere rinsed with acetone and dried. The slides were stored at 4° C.

Example Aldehyde-Silane Coating

The aldehyde-groups were obtained from epoxy-activated slides describedabove. The epoxy group could be broken at acid pH or basic pH. Therupture of the oxirane ring resulted in a diol that was able to oxidizeto aldehyde groups.

Use of Acidic pH

The epoxy-activated slides were incubated with a solution of sulfuricacid at pH 2 for 1 hour at room temperature. Next, the slides wererinsed with water and incubated with 100 mM sodium periodate solutionfor 2 hours at room temperature. The slides were then rinsed with waterand dried. The slides were stored at 4° C.

Use of Basic pH

The epoxy-activated slides were incubated with a solution of sodiumhydroxide at pH 12 for 1 hour at room temperature. Next, the slides wererinsed with water and incubated with 100 mM sodium periodate solutionfor 2 hours at room temperature. The slides were then rinsed with waterand dried. The slides were stored at 4° C.

Example Elispot Assay

An Elispot Spot assay is used to detect T-cell responses by exposing theT-cell to a specific antigen and monitoring the response. A commonresponse is the release of Interferon gamma (IFN-γ).

An Elispot-NAPPA array can be used to simultaneously detect T-cellresponses to many antigens. A T-cell response to a specific antigen onthe array can be captured by attaching an appropriate capture agent to asurface of the array such that the capture agent detects (e.g., binds)to the response signal. The array is contacted with T cells. The releaseof IFN-γ by the T-cell can be detected by affixing an anti-IFN-γantibody onto the array surface.

Example Preparing Self-Assembled Monolayer on a Gold Surface

Different thiols (aliphatic thiols and PEG-thiols) are suspended in anethanol solution to obtain a concentration of 100 mM. This is a stocksolution, which is diluted with ethanol to obtain a coating solutionwith a concentration of 1 mM. Each slide has a capacity for 300 μL. 200μL of the 1 mM solution is added to the slide, and a coverslide addedfast (to prevent ethanol from evaporating). The slides are incubatedovernight. Next, the slides are rinsed with ethanol and dried with anair can.

Other embodiments are within the scope of the following claims:

1-8. (canceled)
 9. A substrate that comprises a surface that includes asurface modification with a group that selectively binds to nucleicacid, and coding nucleic acids distributed at various locations on thesurface.
 10. The substrate of claim 9, wherein the surface issubstantially planar.
 11. The substrate of claim 9, wherein the surfacefurther comprises a binding agent that recognizes a tag and the codingnucleic acids encode polypeptides that include the tag.
 12. Thesubstrate of claim 9, wherein the surface group is NH₂—[CH2]_(n)—NH₃ ⁺and n is between 2 and
 5. 13. The substrate of claim 9, wherein thesurface group is mono-amino-N-aminoethyl (MANAE). 14-20. (canceled) 21.A substrate comprising: (a) a metallic surface; and (b) an array ofprotein aggregates disposed on the surface.
 22. The substrate of claim21, wherein the surface is gold.
 23. The substrate of claim 21, whereinthe surface is substantially planar.
 24. The substrate of claim 21,wherein the aggregates are formed by crosslinking soluble proteins priorto disposing the proteins on the surface. 25-30. (canceled)